Application of Reducible Covalent Capture Purification for Resolving Persulfidome and Nucleolin S-Sulfhydration

Area of Science:

• Biochemistry
• Molecular Biology
• Redox Biology

Background:

• Protein S-sulfhydration is a crucial redox modification regulating protein function.
• Understanding the persulfidome aids in exploring diverse redox regulations.
• Existing methods for persulfidome analysis have limitations.

Purpose of the Study:

• To design and validate a reducible covalent capture method for isolating S-sulfhydrated proteins.
• To analyze the persulfidome in biological samples.
• To monitor specific S-sulfhydrated proteins and modification sites.

Main Methods:

• Development of a reducible covalent capture technique.
• Application of the method to biological samples for persulfidome analysis.
• Enrichment of S-sulfhydrated peptides for site determination.

Main Results:

• Successfully applied the method to reveal S-sulfhydration levels of key proteins like 3-phosphoglyceraldehyde dehydrogenase, NFκB/p65, and nucleolin.
• Demonstrated the technique's utility in enriching S-sulfhydrated peptides.
• Showcased that S-sulfhydration of nucleolin at C543 correlates with nuclear translocation and regulation of downstream targets, impacting oxidative stress response.

Conclusions:

• The developed method effectively isolates and analyzes S-sulfhydrated proteins and peptides.
• This technique facilitates the study of redox regulation by S-sulfhydration.
• The findings highlight the role of nucleolin S-sulfhydration in cellular responses to oxidative stress.